Georges Ducreux a, Darasinh Sihachakr a,*a Morphogenese Vegetale Experimentale, Bat. 360, Uni6ersite Paris Sud, 91405 Orsay Cedex, France
b Research Institute for Food Crops Biotechnology, Bogor 16610, Indonesiac Laboratoire d’Ecologie, Systematique et E6olution, UPRESA-CNRS 8079, Bat. 360, Uni6ersite Paris Sud, 91405 Orsay Cedex, France
d Groupe Endotoxines, UMR 8619, CNRS-UPS, Bat 430 Uni6ersite Paris Sud, 91405 Orsay Cedex, France
Received 3 August 2000; received in revised form 15 September 2000; accepted 15 September 2000
Bacterial wilt, caused by Ralstoniasolanacearum, is one of the most severe diseases ofeggplant (Solanum melongena, 2n=24), especiallyin tropical regions. Perpetuated in soil, it entersthe plant through the roots and progressively in-vades the stem vascular tissues, leading to a partialor complete wilting. It causes heavy yield lossesranging from 50 to 100% [1], thus seriously limit-ing the extensive development of eggplant cultiva-tion. Since agro-chemicals are not effective and
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sanitary cropping systems difficult to apply, con-trol strategies of disease resistance have so farmainly consisted in plant breeding. Althoughscreenings of eggplant accessions were conductedto find sources of resistance [1] and despite the factthat some resistant varieties have been developed[2–5], the level of resistance has become insuffi-cient in hot planting season or poorly drainedfields [6]. Traits of resistance against bacterial wilthave been identified in different wild relatives ofeggplant, such as Solanum tor6um, S. sisymbri-folium [3] and Solanum aethiopicum [3,7]. The lat-ter species can sexually be crossed with eggplant,but the resulting F1 hybrids are either sterile orpoorly fertile, limiting their further use in eggplantbreeding programmes [8,9]. Consequently, thesesexual F1 hybrids were used only as rootstocks foreggplant and tomato in naturally infected fields[8].
The ability of eggplant to regenerate easily fromcultured protoplasts has led to the application ofsomatic hybridization to overcome sexual incom-patibilities and introduce resistance traits fromwild species into the cultivated eggplant [10]. So-matic hybrids of eggplant with Solanum sisymbri-folium were shown to be resistant to nematodesand tolerant to mites [11]. Somatic fusion was alsosuccessfully used to transfer resistance traitsagainst Verticillium wilt from S. tor6um into egg-plant [10–13]. Highly fertile hybrids were recov-ered after somatic fusion between S. melongena cv.Dourga, and one accession of S. aethiopicum [9],but no information has so far been available abouttheir resistance against bacterial wilt.
In this study, in order to further exploit thepotential of bacterial resistance of S. aethiopicum,somatic hybridization was performed by usingprotoplasts fusion between S. melongena cv.Dourga and two accessions of this species. So-matic hybrids were morphologically and molecu-larly characterized, and evaluated for fertility andresistance to bacterial wilt in the field conditions inIndonesia.
2. Materials and methods
2.1. Plant materials
Seeds of eggplant, S. melongena L. cv. Dourga(white half-long fruit), and accessions of S.
aethiopicum, groups aculeatum and gilo, were ob-tained from the collection of I.N.R.A.-Montfavet(France). They were initially sown in vitro, and theresulting plantlets micropropagated by subcultureof leafy node cuttings on modified MS medium[14] containing 20 g l−1 sucrose and solidified with7 g l−1 agar. Vitamins [15] were used at 1/2 andfull strength for eggplant and its wild relativesrespectively. Cultures were kept at 27°C, 60% rela-tive humidity, and 12 h day−1 illumination at 62mmol m−2 s−1. Plants were subcultured at 4–5week intervals.
2.2. Isolation, culture and fusion of protoplasts
About 500 mg of leaves taken from in vitroplants, aged 4 weeks, were scarified and incubatedovernight at 27°C, in 5 ml enzyme solution com-posed of CPW salts [16], 0.5% (w/v) Cellulase RS,0.5% (w/v) Macerozyme R10 (Yakult, Tokyo,Japan), 0.5 M mannitol and 0.05% (w/v) MESbuffer, pH 5.5. After digestion in the dark, proto-plasts were filtered through metallic sieves (100 mmmesh), and then purified and washed by centrifu-gation in 0.6 M sucrose and 0.5 M mannitol+0.5mM CaCl2 solutions successively. Prior to fusion,the density of protoplasts from both species wasadjusted to 3.5×105 protoplasts ml−1.
Electrical fusion experiments were performed asdescribed in Sihachakr et al. (1988) [17]. Briefly,the movable multi-electrodes were placed into a15×50-mm Petri dish containing 600–800 ml of amixture (1:1) of protoplasts from both fusion part-ners. Protoplasts were aligned for 15 s by theapplication of an AC-field at 230 V cm−1 and 1MHz; subsequently, 2 DC pulses developing 1250V cm−1 for 45 ms each were applied to induceprotoplast fusion. The AC-field was then progres-sively reduced to zero. The whole fusion procedurewas followed under an inverted microscope. Afterelectrical treatments, electrodes were removed, and6 ml of culture medium were added progressivelyto the Petri dish containing the mixture of fusedprotoplasts. The culture medium was KM medium[18] supplemented with 250 mg l−1 PEG, 0.2 mgl−1 2,4-D, 0.5 mg l−1 zeatin, 1 mg l−1 NAA, 0.42M glucose as osmoticum and 0.05% (w/v) MES.The pH of the medium was adjusted to 5.8 priorto sterilizing by filtration (0.22 mm filter, Mil-lipore). Cultures were kept in darkness for 7 days,afterwards they were exposed to light. On day 15,
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cultures were diluted eight times with fresh KMmedium supplemented with 2 mg l−1 BAP and 0.1mg l−1 2,4-D. Calli (3–4 mm diameter) were thentransferred to the regeneration medium, composedof MS medium supplemented with vitamins [15],20 g l−1 sucrose, 2 mg l−1 zeatin, 0.1 mg l−1 IAAand solidified with 7 g l−1 agar.
Shoots were excised from callus and multipliedby subculturing leafy node cuttings on hormone-free MS medium. In vitro environmental condi-tions were 12 h day−1 illumination at 62 mmolm−2 s−1, 27°C and 60% humidity.
2.3. Determination of ploidy le6el
The determination of the ploidy level of thehybrids was performed according to Sgorbati et al.[19] with modifications. About 1 cm2 leaf materialfrom in vitro plants was chopped with a razorblade in 1 ml buffer containing CPW salts [16], 0.5M mannitol, 0.25% (w/v) PEG, 0.5% (v/v) TritonX-100, 0.25% (v/v) mercaptoethanol at pH 6.5–7.0. Crude samples were filtered with a nylon net(40-mm mesh) and stained with DAPI at 5 mgml−1. DNA analysis was performed on aPARTEC CA II flow cytometer (Chemunex,Maison — Alfort, France) equipped with a 100-Wmercury lamp (type HBO). Blue fluorescence at455 nm was recorded as a function of relativeDNA content. About 10 000 nuclei were analyzedto generate each histogram. The DNA distributionwas analyzed with DPAC software. The fluores-cence scale was calibrated by using the diploidparental plants as external references.
Cytological analysis was done on root tips takenfrom greenhouse-grown plants as described in Si-hachakr et al. [17].
2.4. Isozyme analysis
Samples of 100 mg of fresh leaves from invitro-grown plants were ground at 4°C in 1.5 ml ofTris–HCl buffer (0.2 M, pH 8.5), containing 20%(w/v) sucrose, 0.03% (v/v) mercaptoethanol, 0.4%(w/v) sodium thioglycolate, 0.4% (w/v) PEG and4% (w/v) polyvinylpyrrolidone (PVP). The mix-tures were centrifuged twice at 15 000×g for 15min at 4°C and the supernatants stored at −80°C.Three systems of isozymes were examined: isoci-trate dehydrogenase (Idh; EC.1.1.1.44), phospho-glucomutase (Pgm; EC.2.7.5.1), 6-phospho-
gluconate dehydrogenase (6-Pgd; EC.1.1.1.43).Isozymes patterns were obtained after elec-trophoresis on 10% starch gels and staining ac-cording to Shields et al. [20].
2.5. Molecular analysis
2.5.1. RAPDsTotal DNA was extracted from in vitro-grown
plants using the DNeasy plant mini kit (Quiagen).Twenty decamer oligonucleotide primers from thekit AB-0320-1 (Fisher) and 14 primers previouslyused on potato by Xu et al. [21] and Baird et al.[22] were tested. PCR reactions contained 30 ngDNA in 25 ml containing buffer (10 mM Tris–HClpH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% TritonX100, 0.2 mg ml−1 gelatin), 0.2 mM of eachdNTP (Genaxis), 20 ng of primer and 1 U of TaqDNA polymerase (Appligene). Amplification wasperformed in a Techne Touchgene Thermocyclerfor 45 cycles. After initial denaturation for 5 minat 92°C, each cycle consisted of 1 min at 92°C, 1min at 37°C and 1 min at 72°C. The 45 cycles werefollowed by a 8 min final extension at 72°C.Amplification products were resolved by elec-trophoresis in 1.4% agarose gel with TBE bufferfor 3 h at 100 V and revealed by ethidium bromidestaining. Gels were photographed on an UV boxwith Polaroid 665 films.
2.5.2. Chloroplast microsatellitesChloroplast patterns were obtained with a pair
of SSR primers designed from Nicotiana tabacumchloroplast sequences [23]: forward primer orSSR-ct1: CGT CGC CGT AGT AAA TAG GAGand reverse primer or SSR-ct1bis: GAA CGTGTC ACA AGC TTA CTC. PCR amplificationof chloroplast microsatellites was performed withthe reaction as described above, primers excepted.The thermal cycling profile was that of Brian et al.[24] including: 5 min at 92°C followed by 45 cyclesof 92°C for 1 min, annealing temperature for 1min, 72°C for 1 min and a final extension for 8min at 72°C. Amplified microsatellites sequenceswere analyzed by electrophoresis, first on 1.8%(w/v) agarose gels containing ethidium bromide,for 3 h at 100 V, then on 6% polyacrylamide-8 Murea denaturing gels for 2 h at 40 W. Before beingloaded on to polyacrylamide gels, the PCR prod-ucts were denatured by incubation at 92°C for 5min in presence of one volume of loading solution
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containing 95% formamide. DNA bands on thepolyacrylamide gels were visualized by silver stain-ing with a silver-sequence DNA sequencing kit(Promega).
2.6. Morphological and fertility analysis
Fourteen somatic hybrid clones and theirparental lines (S. melongena cv. Dourga, S.aethiopicum gr. aculeatum and gilo) were evaluatedfor morphology and fertility in the field conditionsat Cipanas Experimental Station, located in theregion of Bogor (Indonesia). The evaluation wasmade on 30 plants per clone, distributed in threereplicates.
Pollen viability was evaluated by staining pollengrains with fluorescein diacetate (5 mg ml−1). Sam-ples of at least 250 pollen grains each were ob-served under UV light. Viability was expressed asthe percentage of pollen grains with a fluorescentcytoplasm.
2.7. Tests for bacterial resistance
The tests for bacterial resistance were performedin the field conditions at Cipanas ExperimentalStation (Bogor, Indonesia). One strain of R.solanacearum, T 926 (race 1, biovar 3), isolatedfrom Solanum tor6um, and kindly provided by theResearch Institute for Spice and Medicinal Crops(Bogor, W. Java, Indonesia), was used. Virulentcolonies of R. solanacearum were selected after 2days at 28°C on tetrazolium chloride medium(TTC) [25]. Bacteria were then routinely grown onsucrose-peptone medium (SPA) at 28°C [26]. Be-fore inoculation, bacterial cells suspensions wereprepared in sterile distilled water, using 1-day-oldcultures, and their concentration was adjusted byspectrophotometry to 107 cfu (colony formingunits) per ml (OD650=0.1).
Vitroplants were transplanted into individualplastic pots containing a sterilized mixture of soiland manure (1:1) and grown in the greenhouse for4 weeks. Thirty healthy and uniform plants perclone, distributed in three replicates, were inocu-lated by stem pricking: after wiping the base of thestems with a tissue paper soaked in 70% ethanoland smearing it with Vaseline®, the calibratedbacterial suspension was pricked into the tissueswith sterilized needles. Control plants were inocu-lated using sterile distilled water. One day later, allthe plants were transplanted to the field.
Two, 4 and 8 weeks after inoculation, diseaseintensities were scored by using a foliar symptomscale ranging from 0 to 5: 0, healthy plants; 1, upto 25% wilted leaves; 2, up to 50% wilted leaves; 3,up to 75% wilted leaves; 4, up to 100% wiltedleaves; and 5, dead plants. A disease index wascalculated for each clone according to Winsteadand Kelman [27]: DI= [(sum of scores)/(N×max-imum score)]×100, with N being the number ofinoculated plants per clone.
Data on disease evaluation were subjected tostatistical analysis using a fixed model of analysisof variance (ANOVA) with two criteria of crossclassification: effects of genotype and period ofassessments. Means separation was done by usingDuncan’s multiple-range test [28].
3. Results
3.1. Production of somatic hybrids
As described in Sihachakr et al. [17,29] andDaunay et al. [9], and in contrast to the resultsreported by Jarl et al. [13], leaves from in vitroplants were a competent source of viable proto-plasts giving approximatly 4×106 cells g−1 freshmaterial. Several successful fusion experimentswere conducted with fusion frequencies rangingfrom 10 to 20%. Two weeks after dilution of thefusioned protoplast suspension, hundreds of mi-crocolonies appeared, and rapidly developped intocalli when transferred onto a solid growthmedium. Putative somatic hybrid calli were se-lected according to their ability to grow faster andto regenerate earlier than the parents [10]. There-fore, 2 weeks later, only those of at least 2–3 mmin size were transferred onto the regenerationmedium. After 5 weeks, about 9% of the 950selected calli produced shoots. One to three shootswere excised from each regenerating callus andsubcultured on hormone-free MS medium. Fi-nally, 83 calli gave rise to 120 plants, of which 82from the fusions between Dourga and S.aethiopicum gr. aculeatum, and 38 from the fusionsbetween Dourga and S. aethiopicum gr. gilo.
3.2. Ploidy le6el
The ploidy level of the regenerated plants wasdetermined by using flow cytometry. The position
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Fig. 1. Histograms of relative nuclear DNA contents obtained by flow cytometric analysis of 10 000 DAPI-stained nuclei isolatedfrom leaves of the dihaploid parents: S. melongena cv Dourga (A), S. aethiopicum gr. aculeatum (B) and gr. gilo (C), and of twoof their tetraploid somatic hybrids: DSa-18a (D) and DSa2-2 (E). Fluorescence intensity is proportional to nuclear DNA quantityand the position of the dominant peak reflects the ploidy level.
of dominant peaks corresponding to nuclei atG0–G1 phase of the cell cycle, was comparedbetween putative hybrid and parental plants (Fig.1). The analysis revealed that 23 out of 82 plantsregenerated from the fusion between Dourga andS. aethiopicum group aculeatum, referred as toDSa, and seven out of 38 plants recovered fromthe fusion between Dourga and S. aethiopicumgroup gilo, referred as to DSa2, were at the ex-pected tetraploid level. They were retained forfurther analyses to confirm their hybridity. Theremaining plants, representing about 75% of thetotal, were diploids.
Chromosomal countings made on root tips of arandom sample of hybrids confirmed the resultsobtained by flow cytometry, the tetraploid hybridsshowing 2n=4x=48 chromosomes per metapha-sic cell (Fig. 2). Because of their morphologicalsimilarity, the two chromosome sets could not bedistinguished from each other.
3.3. Isoenzyme analysis
Three isoenzyme systems, Pgm, 6-Pgd and Idh,
were examined to confirm hybridity of the 30selected tetraploid putative hybrids. They revealedpolymorphism between the parental lines and dis-tinguished the hybrids from the parents. For Pgm,the somatic hybrid patterns contained bands iden-tical to the mixed extracts of the parents (Fig. 3B).
Fig. 2. Root-tip metaphasic cell of a tetraploid somatic hybrid(DSa 110) between S. melongena cv Dourga and S.aethiopicum gr. aculeatum (2n=4x=48 chromosomes).
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Fig. 3. Electrophoresis banding patterns of (A) 6-phosphoglu-conate dehydrogenase (6-Pgd), (B) phosphoglucomutase(Pgm) and (C) isocitrate dehydrogenase (Idh). Line D: S.melongena cv Dourga; line Sa: S. aethiopicum (both groupsaculeatum and gilo had the same pattern); line M: mixture ofDNA from S. melongena cv Dourga and S. aethiopicum ; lines1–7: hybrids DSa 1a, DSa 3a, DSa 4a, DSa 6a, DSa 17, DSa20a, and DSa 26a; lines 8 and 9: hybrids DSa2-2 and DSa2-3.
14, 16, 17, 18, 20) and three primers previouslyused on potato (A10, A12, SC10-01) revealedpolymorphism between the two pairs of parents.Five of them, AB1-0320-1/07, 08, 10, 12 andSC10-01, showing the best diagrams were chosento analyze the hybrids.
AB1-0320-1/08 (Fig. 4A), AB1-0320-1/12 andSC10-01 (not shown) led to hybrid profiles withspecific bands of both parents, thus confirming thehybridity of the 30 selected plants. For all theseplants, the patterns obtained with AB1-0320-1/10
Fig. 4. Electrophoresis banding patterns of PCR amplificationproducts. (A), (B), (C) RAPDs patterns obtained on 1.4%agarose gels using the primers AB1-0320-1/08, 10 and 07respectively. (D) Chloroplast microsatellite patterns obtainedon a 6% polyacrylamide gel using the couple of primersSSR-ct1/1bis. Line D: S. melongena cv Dourga; line Sa: S.aethiopicum (both groups aculeatum and gilo had the samepattern); line M: mixture of DNA from S. melongena cvDourga and S. aethiopicum ; lines 1–7: hybrids DSa 1a, DSa3a, DSa 4a, DSa 6a, DSa 17, DSa 20a, and DSa 26a; lines 8and 9: hybrids DSa2-2 and DSa2-3.
For 6-Pgd and Idh, in addition to the sum of theparental bands, the hybrid pattern showed anadditional band that was specifically relevant tothe hybrid nature of the plants tested, and notfound in the parental mixed extracts (Fig. 3A andC).
3.4. Nuclear genome analysis
The nuclear genome of the tetraploid putativehybrids was analyzed by PCR using 34 RAPDs,four SSR and two Inter-SSR primers. All therandom primers used generated PCR productsfrom the genomic DNA of both parents. Theyresulted in the amplification of two to 12 DNAfragments, from 0.1 to 1.2 kb. Fifteen primers ofthe kit (AB1-0320-1/1, 2, 4, 6, 7, 8, 10, 11, 12, 13,
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Table 1Plant height, stem diameter, number and length of branches, length and width of leaves (Means of 30 plants 9S.D.)a
a The hybrids are designated by a number identifying the callus from which they are derived and a letter (a, b, c) when severalplants are from the same callus.
(Fig. 4B) and AB1-0320-1/07 (Fig. 4C) showedonly specific bands from Dourga, and from S.aethiopicum, respectively. When combined to-gether, these results constitute a supplementaryproof, though indirect, of the hybrid nature of the30 tetraploid plants.
3.5. Chloroplast genome analysis
The ct genome type of the hybrids was deter-mined by PCR using the couple of primers SSR-ct1/1bis. The amplification products were of aboutthe expected length of 89 pb, as measured bycomparison to the 100 bp DNA ladder (Biolabs)and allowed to distinguish the chloroplastgenomes of the parents. All the hybrids showedthe pattern of either one parent or the other (Fig.4D). Among the 23 DSa hybrids, 14 possessed theS. melongena ct type and nine that of S.aethiopicum gr. aculeatum. The distribution of ctDNA was similar among the seven DSa2 hybrids:
four and three with S. melongena and S.aethiopicum gr. gilo ct type respectively.
3.6. Morphological and fertility analysis
Fourteen somatic hybrid clones, including 12DSa, two DSa2 and their respective parents, wereevaluated for morphology and fertility in fieldconditions at the Cipanas Experimental Station(Bogor, Indonesia). As shown in Table 1, thehybrids grew vigorously and were taller than theparental lines. Their stem diameter was lower orintermediate between the parents. Their numberand length of branches, as well as their length andwidth of leaves were rather close to those of thewild parents. The shape of hybrid leaves, flowersand fruits was relatively homogeneous and inter-mediate between the parents (Fig. 5). The somatichybrids produced more flowers than the parents,but many of them aborted, and only few gave riseto fruit production (Table 2). Pollen viability,
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measured by staining pollen grains with fluores-cein diacetate, ranged from 28 to 50% for DSahybrids and 37 to 40% for DSa2 hybrids, whereasthe parental plants had 60–65% viable pollen. Allhybrids set fruits with viable seeds. Traits of hy-brid fertility, including the number, size andweight of fruits, was intermediate between theparents, except for two hybrid clones, DSa 6a andDSa 16 with less fruit production (Fig. 5, Table 2).
Taking into account the intermediate morphol-ogy, the ploidy level and the analysis of nuclearand chloroplast genomes of the selected plants byexamining the isozymes and DNA markers, the 30selected plants were confirmed to be somatic hy-brids between S. melongena and S. aethiopicum.
3.7. Tests for resistance to bacterial wilt
For reasons of safety and limitation of pathogenspreading, the stem pricking inoculation methodseemed to be the most appropriate for field trials.However, in order to determine the inoculationimpact on evaluation of hybrid resistance to R.solanacearum, preliminary experiments were con-ducted in the greenhouse to compare differentmethods of bacterial inoculation: stem pricking,
soil drenching and root immersing. Three sets ofplants were inoculated at the same time. Stempricking was carried out as described previously.Soil drenching consisted in adding 40 ml of bacte-rial suspension around the roots slightly woundedwith a knife without digging up the plants. Forroot immersing, the plants were removed fromtheir pots, and their roots were dipped into thebacterial suspension for 30 min before they werereplanted. As for pepper [30] and unlike tomato[31], preliminary results showed that the wiltingscores obtained for the three sets of plants werenot significantly different (data not shown) andthe inoculation methods did not seem to affect theevaluation of the resistance levels. Therefore, onlythe stem pricking technique was used for furtherbacterial tests.
Eighteen somatic hybrid clones, including 16DSa and two DSa2, derived from separate calli,and their respective parental lines, were evaluatedfor resistance to R. solanacearum (race 1, biovar3). The tests were performed from the end of therainy season, in the fields of the Cipanas Experi-mental Station, located to the south of Bogor, atan altitude of 900 m above the sea level, in one ofthe main eggplant cultivation areas in Indonesia.
Fig. 5. Flowers from S. melongena cv Dourga (A), S. aethiopicum gr aculeatum (C), and their somatic hybrid (B). The white linein (C) is the scale for (A), (B) and (C). Fruits from S. melongena cv Dourga (D), S. aethiopicum gr aculeatum (F), and theirsomatic hybrid (E). The white line in (E) is the scale for (D), (E) and (F).
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Table 2Number of flowers per plant, number of fruits per plant, % of flowers setting fruit, fruit mean weight and fruit yield per plant(Means of 30 plants 9S.D.)a
Nb Nb Weight of fruitsct DNA type % flowers givenLinesfruits/plantflowers/plant rise to fruits
g fruit−1 g plant−1
D 11.3390.22 5.8090.61S. melongena cv 49.6994.43 254.3890.79 1468.109151.31Dourga (D)
Sa 64.7395.26 23.2791.48S. aethiopicum gr. 37.8491.40 21.7690.34 503.61930.77aculeatum (Sa)
S. aethiopicum gr. gilo Sa2 127.7593.23 14.5490.66 8.4490.32 44.4190.34 493.54933.57(Sa2)
a The hybrids are designated by a number identifying the callus from which they are derived and a letter (a, b, c) when severalplants are from the same callus. Ct DNA type of S. melongena cv. Dourga (D), S. aethiopicum (Sa).
The cultivated eggplant, cv. Dourga, was suscepti-ble, showing bacterial wilt symptoms (necrosis andwilting) on lower leaves 1 week after inoculation.All plants of eggplant died within 2 weeks, withdisease indices of 100. Both groups aculeatum andgilo of the wild species, S. aethiopicum, displayedsimilar level of resistance against bacterial wilt,with only 50% wilted leaves on average (Table 3).The ANOVA of disease indices showed highlysignificant effects of the period of assessment andgenotype on response to bacterial wilt at P=0.01,but no significant effect of interaction betweenthese two criteria was observed at P=0.05. Inorder to compare the resistance levels of the hy-brids and the parents, means of disease indices onthe three periods of assessment were calculatedand classified according to Duncan’s multiple
range test (Table 3). The level of resistance of thewild species was significantly higher than that ofthe susceptible cultivated eggplant, with 51.9 and100% wilted leaves respectively. Eight DSa hybridsand two DSa2 hybrids appeared to have levels ofresistance similar to those of S. aethiopicum, withmeans of disease indices ranging from 29.3 to 50.4(Table 3). Interestingly, six DSa hybrids were sig-nificantly more resistant to race 1 strain than thewild parent, S. aethiopicum, with means rangingfrom 23.3 to 27.8 (Table 3). The disease indices ofmost hybrids and the wild species decreased withthe period of assessment (Table 3), indicating thatless leaves wilted 8 weeks after inoculation. In fact,those genotypes were tolerant to bacterial wilt,and leaves that had newly been formed did notwilt as the plants grew up.
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4. Discussion
In this study, somatic hybrids have successfullybeen recovered after electrical fusion between pro-toplasts from S. melongena and two accessions ofS. aethiopicum. Early selection of putative hybridsbased on differences in cultural behavior of calli,was effective since 25% of selected plants wereconfirmed to be somatic hybrids. Similar methods,exploiting hybrid vigor as the only selection sys-tem of the hybrid calli, were previously used toobtain somatic hybrids of eggplant [10] and potato[32,33].
The early characterization of regenerated plantsincluded determination of their ploidy level byflow cytometry, and confirmation of their hybridstatus by isoenzymes and RAPDs analyses. Mor-phological observations and chromosome count-ing were conducted afterwards on plants grown tomaturity in the greenhouse and the field, andconfirmed the hybrid nature of the selected plants.
RAPD markers were a powerful tool to character-ize the nuclear genome of the hybrids. As theywere expressed as dominant, the presence of atleast one polymorphic amplification product fromeach parent in the patterns of the tested plants wasenough to prove their hybridity. However, with afew of the 34 oligonucleotides tested, all the regen-erated plants, and also the mixture of both parentsDNA, showed the bands from only one parent.The same observations were previously reported insomatic hybrids between S. tuberosum and S. bre6-idens [34] and in several DNA mixing experimentsreviewed in Reineke et al. [35]. The reasons of thisphenomenon are unclear. It could be due to com-petition effects on primer-binding sites in thegenome [36], especially if the RAPD productsshare extensive sequence homologies [35]. Thepresence of repetitive sequences in the RAPDsproducts could also lead to the suppression of theamplification by the formation of heteroduplexesbetween different copies of these repetitive se-
Table 3Disease indices (DI) recorded 2, 4 and 8 weeks after root inoculation by race 1 strain of R. solanacearuma
Lines Periods of assessment Means
8 weeks4 weeks2 weeks
100.0090.00 100.0090.00 100.0090.00 100 aS. melongena cv Dourga (D)59.3394.56 49.3394.87S. aethiopicum gr. aculeatum (Sa) 46.6795.57 51.8 b
49.4191.77 46.7491.67 51.9 bS. aethiopicum gr. gilo (Sa2) 59.4492.13
DSa-18a 46.6791.2351.3390.91 53.3394.59 50.4 bDSa-1a 45.3391.8049.3393.37 40.0091.82 44.9 bc
39.6791.32DSa2-3 34.0090.39 33.3392.10 35.7 bc41.3391.64DSa2-2 44.0090.36 33.3391.05 39.6 bc
a Disease intensities were scored using a 0–5 foliar symptom scale: 0, healthy plants; 1, up to 25% wilted leaves; 2, 26–50% wiltedleaves; 3, 51–75% wilted leaves; 4, 75–100% wilted leaves; and 5, dead plant. DI= [(sum of scores)/(N×maximum score)]×100,where N is number of tested plants per clone. ANOVA was performed with two criteria of cross classification (genotype andassessment period): highly significant effects of the genotype (F(df: 17; 108)=13.5) and the assessment period (F(df: 2; 108)=13.4) atP=0.01, and non significant effect of interaction (F(df: 34; 108)=0.5) at P=0.05. Significant difference between genotypes atP=0.05 is indicated by different small letters.
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quences [35]. In these cases, it is not one primer,but the combination of a couple of primers, one,allowing the amplification of specific bands fromone parent, and the other, the amplification ofspecific bands from the other parent, which consti-tutes a proof of the presence of both genomes inthe hybrid.
In most somatic hybridization experiments deal-ing with different species, the initial mixture of thetwo parental cytoplasms was followed by the elim-ination of one of the two parental ctDNAs [37].This was also the case for each hybrid recovered inthis study. They all showed chloroplasts from onlyone parent. Biased chloroplast segregations werefrequently reported in somatic hybrids [9,12]. Thereason of this phenomenon could be a differencein organelle replication rate. But in this study, asreported in Pehu et al. [34] and in San et al. [37],the segregation of chloroplasts did not seem to bebiased in favor of one or the other parent, since 14out of the 23 DSa hybrids and four out of the 7DSa2 had the eggplant ctDNA type, and the restthat of S. aethiopicum. In contrast to Daunay etal. [9], no correlation between the ctDNA type andany of the hybrid fertility criteria was observed inthis study. The ctDNA type was not correlatedeither to the different resistance levels expressed bythe hybrids.
Hybrid fertility is a prerequisite for further ex-ploitation of somatic hybrids in breeding pro-grams. Attempts at overcoming sexual barriersbetween distant relatives and eggplant throughsomatic hybridization often resulted in sterile hy-brids [10]. This was observed with S. sisymbri-folium [11], S. khasianum [17], S. nigrum [38] andS. tor6um [12,29]. This frequent sterility could bedue to incompatibility between distantly relatedgenomes. However, somatic hybrids between dis-tant relatives may show reasonable levels of femalefertility, as observed in backcrosses betweenpotato and somatic hybrids of potato with S.bre6idens, used as female parent. In the mean time,the hybrid pollen was ineffective in pollinations[39]. Likewise, fertility was recovered in highlyasymmetric hybrids of eggplant with S. tor6um[13]. When the fusion partners are phylogeneticallycloser, as S. melongena and S. aethiopicum, theirprogeny is more likely to be fertile. Our results onfertility of somatic hybrids of S. melongena with S.aethiopicum confirm those already obtained byDaunay et al. [9], and strongly support thishypothesis.
In the environmental conditions of our tests, allthe somatic hybrids showed a similar or higherlevel of resistance to R. solanacearum race 1, bio-var 3 than S. aethiopicum, both groups gilo andaculeatum being tolerant. From 2 to 8 weeks afterinoculation, the disease indices of some of thehybrids progressively decreased, as new leaveswere developing at the top of the plants. Thisapparent moving of the symptoms to the lowerparts of the plants could be due to a limitation ofspread of the bacteria. Tolerance to bacterial wiltin tomato was reported to be linked to a coloniza-tion restricted to vascular tissues [40], and wasattributed to the induction of nonspecific physicalbarriers like the production of tyloses and otherdeposits [41].
In this study, interestingly, six hybrids weresignificantly more tolerant to bacterial wilt thanthe tolerant parent. If they did not undergo fluctu-ations of the experimental conditions in the field,these results could be linked to hybrid vigor andmay be due to superdominance phenomenons re-sulting from genetic interactions induced by thecombination of the two parental genomes. In fact,those somatic hybrids were among the most vigor-ous and productive clones that had been obtainedin this study (Tables 1 and 3).
Results from this study are very encouragingand demonstrate that somatic hybridization is arapide and effective way to introduce new sourcesof resistance into eggplant. The somatic hybridsobtained showed a similar or even higher level ofresistance to R. solanacearum race 1 than theresistant parent. Nevertheless, further studies areneeded to evaluate the stability of this resistanceunder different temperature and field conditions.Race 1 is a highly heterogeneous group commonin the low-land tropics that can cause disease onmany different species [42]. So far no data onstrain specificity of the S. aethiopicum wilt resis-tance have been available and it would be interest-ing to test the resistance of the hybrids againstsome of the four other races of R. solanacearum,such as race 3, which is also very common in Asia.
By using anther and microspore culture, fertiledihaploid progenies have already been obtainedfrom several somatic hybrids recovered in thisstudy (data not shown). In order to produce newmarketable varieties resistant to bacterial wilt,they are being used as breeding materials in back-crossing to the diploid recurrent eggplant includ-
C. Collonnier et al. / Plant Science 160 (2001) 301–313312
ing resistance tests at each generation. Resistanceto bacterial wilt in tomato was reported to bepolygenic, and some QTL were detected on chro-mosomes 4 and 6 [43,44]. In eggplant, resistanceseems to be controlled by a single dominant gene[45] and by a more complexe mixture of partiallydominant or recessive genes [46]. Further analysisof selfed and backcrossed progenies of the dihap-loids we obtained would contribute to elucidatethe nature and inheritance of the resistance to R.solanacearum. These plants may also be useful todetermine molecular markers linked to the resis-tance, through Bulk segregant analysis (B.S.A.)for example.
Acknowledgements
The authors thank the EU (contract No. IC18-CT97-0187) for financial support.
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